Probe optimization methods

Abstract

The present invention provides methods for optimizing nucleic acid detection assays for use in basic research and clinical research. Specifically, the invention provides a method for empirically optimizing nucleic acid probes by testing them with samples containing genomic DNA with variations in copy number in different regions of the genome. The invention enables the optimization of probes for any hybridization based assay including microarrays, bead-based assays, genotyping assays and RNAi assays.

1 . A method for optimizing nucleic acid probes used to identify at least one genetic alteration in a test genome, the method comprising the steps of: 
 a) providing a genomic sample mixture comprising a test genomic sample and a reference genomic sample, wherein the test genomic sample has genetic alterations;    b) labeling the genomic sample mixture;    c) hybridizing the labeled genomic sample mixture to a microarray designed to have probes that hybridize to the region of known genetic alteration, such that an intensity pattern is produced, wherein the hybridization step is performed at least one time; and    d) selecting at least one optimized probe corresponding to the region of known alteration in the test genome, wherein the probe exhibits a signal intensity which best reveals a genetic alteration in the test genomic sample relative to the reference genomic sample.    
   
   
       2 . A method as claimed in  claim 1  wherein the method also includes making subsequent microarrays including the optimized probe therein.  
   
   
       3 . The method of  claim 1  further comprising the step of: 
 e) identifying at least one genetic alteration in the test genome.    
   
   
       4 . The method of  claim 1  wherein the genetic alteration is an amplification or deletion in a chromosome.  
   
   
       5 . The method of  claim 1  wherein the genetic alteration covers an entire chromosome.  
   
   
       6 . A method for testing for genomic alterations in a target genomic region, the method comprising the steps of 
 a) identifying optimized probes by the steps of 
 (1) obtaining a test genomic sample containing a genomic alteration and a reference genomic sample without the genomic alteration;  
 (2) making a microarray with probes designed to hybridize in the region of the genomic alteration;  
 (3) hybridizing the test genomic sample to the microarray and analyzing the nature of the hybridizations; and  
 (4) identifying at least one probe which is best optimized to reveal the genomic alteration;  
   b) making a microarray which contains the optimized probe;    c) hybridizing the microarray against a genomic sample of unknown content; and    d) using the information from the location of the hybridized probe to determine if the genomic alteration is in the genomic sample.    
   
   
       7 . A method as claimed in  claim 6  wherein the genomic alteration is a change in copy number of a genomic segment.  
   
   
       8 . A method as claimed in  claim 6  wherein each of the microarrays is made by a maskless array synthesis instrument.